Drug discovery may benefit from multiparametric analysis of cell cycles

Recent advances in drug discovery may help find improved therapies
in oncology. Researchers are working to develop drugs that can block cell proliferation
and ultimately induce apoptosis by inhibiting cell-cycle machinery. They look for
these drugs by observing how compounds affect cellular mechanisms, using medium-throughput
assays to perform multiparametric analysis of the cell cycle.

Flow cytometry is commonly employed to this end
and has been used to do three-parameter analysis of relevant cell-cycle markers.
But because it can be used only in cell suspensions, obtaining information about
adherent cell cultures can be difficult, if not possible.

Researchers have performed
multiparametric analysis of cell-cycle markers using a system that combines fluorescence
microscopy with image processing, automation and informatics. This could shed light
on how drugs affect cellular mechanisms, and could contribute to improved therapies
in oncology, for example.
Microscopy-based imaging techniques
for multiparametric analysis of cell-cycle phases could circumvent these problems.
In a Journal of Biomolecular Screening paper published Sept. 1, researchers
at Nerviano Medical Sciences in Nerviano, Italy, reported a study in which they
employed a high-content screening technique that enables quantification of multiple
cellular markers at the single-cell level using fluorescent probes and automated
microscopy. They demonstrated four-color analysis with the technique, highlighting
its potential for screening of cell-cycle-specific inhibitors.

“From our point of view, the
possibility of tracking cell-cycle perturbations induced by pharmacologically active
compounds in high-density microplate format, minimizing artifacts, represents itself
an important step forward [for high-content screening],”said Fabio Gasparri,
one of the authors of the study. “This approach would allow determination
of accurate cell-cycle-related information, together with proliferative and morphometric
cellular data, with a minimum consumption of reagents, cells and time with respect
to other cytometric techniques.”

The investigators used the ArrayScan
system made by Cellomics Inc. of Pittsburgh. This fluorescence microscopy imaging
system was designed specifically for high-content screening and analysis, combining
fluorescence microscopy with image processing, automation and informatics to produce
an “industrialized” workstation for scientists investigating cell populations.
The system includes a broad white-light source, a cooled CCD camera, optics by Carl
Zeiss and controller software.

Because the system performs automated
acquisition and image analysis more or less simultaneously, researchers can run
a 96-well plate in a matter of minutes. In addition, said Judy Masucci, the company’s
director of marketing, it adds intelligence to the imaging process. Users can tell
the system to continue imaging until it sees x number of positive or negative
events, for example. They also can enter stop criteria. “If you have a bad
well, with no cells in it, you don’t want to waste time taking images of that
well,” she said. “And if you have five bad wells in a row, you can set
it to throw out the whole plate. That greatly increases productivity.”

Gasparri cites several advantages of
the system, including the user-friendly interface; excellent optics and accessories;
a sophisticated, integrated data storage/analysis platform; and reliable hardware/software
components. “The latter, in particular,” he said, “is a very important
feature for screening purposes involving prolonged running periods.”

As with any reader based on a conventional
fluorescence microscope, he added, there are several general disadvantages with
respect to, for example, laser-scanning cytometers. One of these is the relative
slowness of the system because of autofocusing and serial acquisition of fields.
Another is the comparatively low coefficients of variation for fluorescence distribution
histograms, the result of the lamp being less potent than the lasers used in laser-scanning
cytometers. This leads to difficulty quantifying cell-cycle phases by DNA content
distribution alone. Because of this, the researchers reported percentages from only
one of the cells as a quantitative parameter in the DNA histograms.

Four-color analysis of the cell-cycle markers
yielded a great deal of additional information. For example, combining the markers
in three-dimensional plots revealed five subpopulations that would have been difficult
to analyze by looking at DNA content alone.
Still, the multiparametric analysis
afforded by the system could contribute to significant advances in high-content
screening. Previous methods used for cell-cycle analysis were mostly based on quantification
of DNA content alone, which doesn’t allow for accurate tracking of individual
cell-cycle phases, Gasparri noted. Analysis of DNA content as well as other, independent
cell-cycle markers would allow much more precision — precision proportional
to the number of additional markers included, he said.

The Nerviano group performed four-color
analysis of a cell population to determine DNA content and bromo-2-deoxyuridine
(BrdU) incorporation, cyclin B1 expression and histone H3 phosphorylation (the
latter three indicate cell cycle) following treatment with thymidine, paclitaxel
or nocodazole; they chose these markers because they allow tracking of the main
events of the cell cycle. To avoid crosstalk, they employed primary and secondary
antibodies to separate fluorescence signals based on their emission spectra and
location within cells.

The researchers
analyzed BrdU incorporation, cyclin B1 expression and histone H3 phosphorylation
(p-H3) as well as DNA content (DAPI), following treatment with several compounds.
The additional markers enabled much more precision than would have been possible
analyzing DNA content alone. Reprinted with permission of the Journal of Biomolecular
Screening.
They brought together a variety of
existing approaches to show the efficacy of the method. “The protocols used
in the study were essentially a combination of previously established techniques
that we re-employed to solve practical difficulties encountered in developing a
four-color imaging approach,” Gasparri said. For example, they used nuclease
to denature chromatin, rather than the more common but harsher acid pretreatment,
and thus addressed some of the challenges of quantification BrdU incorporation during
S-phase.

The results correlated well with those
from flow cytometry, demonstrating that they could track blocking of specific cell
cycles induced by treatment with thymidine, paclitaxel or nocodazole, and confirming
the potential of the technique for high-content screening. Gasparri emphasized,
however, that they don’t expect the technique to replace flow cytometry as
the technique of choice for multiparametric analysis of the cell cycle, but rather
to provide a complementary method with which to investigate adherent cell populations
with minimal sample handling.

The researchers plan to continue developing
and validating high-content screening assays to spur acceptance of the technique
in academic research and in industry, and to explore its further possibilities and
limitations. In addition, they are studying various applications of high-content
analysis. For example, they are working to create a database reporting the profile
of the cellular mechanism of action of selected reference compounds as well as a
systematic analysis of siRNA cellular phenotypes.&